Cardiovascular disease is the leading cause of mortality and coronary heart disease alone is responsible for more than half of these deaths. The occurrence of a coronary event is due, in the vast majority of cases, to the rupture of a vulnerable or unstable coronary plaque, resulting in a sudden block of blood flow in critical arteries in the brain, the lungs or the heart. Several of these patients die suddenly of a first myocardial infarction or cardiac arrest without any symptoms or diagnosis of coronary artery disease (Naghavi et al., 2003, Circulation 108:1664-1672). Today, no general diagnostic method is available for detection or characterization of vulnerable plaques. Coronography, the reference method for the diagnosis of coronary artery disease, allows visualization of abnormal reductions of the internal diameter of an artery, called “stenoses,” but does not allow the identification of non-stenotic plaques. Nuclear imaging holds potential for molecular imaging of vulnerable atherosclerotic plaques. Many tracers of various chemical nature, including lipoproteins, peptides, oligopeptides, antibodies, sugars, antisense nucleotides and nanoparticles were evaluated experimentally for molecular imaging of atherosclerosis (Riou et al., 2009, Curr. Med. Chem. 16:1499-1511). The main evaluated targets were oxidized LDLs and their receptors, the inflammatory process via macrophage cell imaging, or imaging of receptors or enzymes expressed by this cell type, apoptotic phenomena and the phenomenon of neoangiogenesis. Among tracers targeting the inflammatory process, 99m Tc-MCP-1 for nuclear imaging via SPECT (Single Photon Emission Computed Tomography) and [18F]-FDG for PET (Positron Emission Tomography) imaging have been used for in vivo noninvasive imaging of macrophage accumulation in experimental atherosclerotic lesions. On a clinical level, [18F]-FDG and 99mTc-Annexin A5 allowed noninvasive imaging of the accumulation of macrophages and apoptotic cells, respectively, in carotid atherosclerotic plaques of symptomatic patients. However, none of these radiotracers is currently used in routine clinical practice, mainly because of their inability to reach sufficient ratios of lesion versus background noise level in the coronary lesions. Indeed, nuclear imaging of vulnerable plaques in the coronary arteries is particularly difficult because of the low volume of the lesions and their proximity to blood that contains circulating unbound tracer.
An ideal tracer should combine high affinity and specificity, good solubility and stability and efficient radiolabeling with small size and fast blood clearance, so that high contrast images can be obtained shortly after administration. Nanobodies constitute a promising new class of radiotracers that might adhere to these conditions. They are derived from unique heavy-chain-only antibodies that are by nature present in camelids and represent the smallest possible (10-15 kDa) functional immunoglobulin-like antigen-binding fragment. Nanobody-based tracers targeting cancer antigens epidermal growth factor receptor, carcinoembryonic antigen, or human epidermal growth factor receptor 2 (HER2) with (sub)nanomolar affinities have already proven their ability to generate highly specific contrast images for non-invasive bio-imaging of cancer cells in mouse tumor models (Huang et al., 2008, Mol. Imaging Biol. 10:167-175; Vaneycken et al., 2010, J. Nucl. Med. 51:1099-1106; Vaneycken et al., 2011, FASEB J. 25:2433-2446). Recently, in hypercholesterolemic ApoE-deficient mice, representing a mouse model of atherosclerosis, it was documented that high contrast images and high lesion-to-heart and lesion-to-blood ratios could be obtained via SPECT imaging using Nanobodies targeting vascular cell adhesion molecule-1 (VCAM-1). (Broisat et al., 2012, Circ. Res. 110:927).